Number Of Immunogold Particles Research Articles

Ultrastructural immunolocalization of laminin 332 (laminin 5) at dento-gingival interface in Macaca fuscata monkey.

Although laminin 332 (laminin 5), an extracellular matrix molecule involved in cell adhesion and migration, has been localized at the interface between the tooth enamel and junctional epithelium, its ultrastructural localization remains to be fully clarified. The purpose of the present study was to investigate the ultrastructural distribution of laminin 332 at the dento-gingival interface in Japanese monkey (Macaca fuscata) using pre- and post-embedding immunoelectron microscopy. Pre-embedding immunoelectron microscopy revealed a broad band of internal basal lamina together with supplementary lamina densa, and both showed immunolabeling for laminin 332. Immunoreaction products for laminin 332 were observed in the rough-surfaced endoplasmic reticulum of the junctional epithelial cells close to the tooth enamel. Post-embedding immunoelectron microscopy revealed an increase in the number of immunogold particles toward the coronal portion, resulting in a large accumulation of particles on the basal lamina, preferentially on the lamina densa. Concomitantly the dental cuticle at the dento-gingival interface was sporadically, but specifically, immunogold-labeled with anti-laminin 332 antibody. These data suggest that junctional epithelium actively produces laminin 332, and that the products accumulate at the dento-gingival interface during cell migration coronally towards the gingival sulcus.

Increased expression of cannabinoid receptor 1 in the nucleus accumbens core in a rat model with morphine withdrawal

Relapse is a major clinical problem and remains a major challenge in the treatment of drug addiction. There is strong evidence that the endocannabinoid system of the nucleus accumben core (NAcc) is involved in drug-seeking behavior, as well as in the mechanisms that underlie relapse to drug use. To reveal the mechanism that underlies this finding, we examined the expression pattern of the cannabinoid receptor 1 (CB1-R) in the NAcc of SD rats that had been undergoing morphine withdrawal (MW) for 1 day, 3 days and 3 weeks (acute, latent and chronic phases, respectively). Morphine exposure induced conditioned place preference (CPP) in rats. Significant increase of CB1-R expression in NAcc was observed in animals in the 1 day, 3 days and 3 weeks morphine withdrawal compare to the control group. Immunofluorescence labeling showed axonal fibers or terminals by fluorescence microscope observation. Immunoelectron microscopy detection showed silver-gold particles located in the presynaptic membranes that mainly give rise to symmetrical synapses. Quantitative electron microscopy showed an increase in number of CB1-R-positive terminals in the morphine withdrawal groups and the number of immunogold particles was significantly increased at these inhibitory terminals. We also confirmed that infusions of the CB1-R antagonist rimonabant into the NAcc attenuated the CPP during morphine withdrawal. Our present data have thus indicated that increasing pattern of CB1-R expression in the NAcc during above morphine withdrawal phases, which might underlie the relapse associated drug seeking behavior after morphine withdrawal.

Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels

P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ∼40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.

Genotoxic effects of chromium(VI) and cadmium(II) in human blood lymphocytes using the electron microscopy in situ end-labeling (EM-ISEL) assay

Evaluation of genotoxic effects of potassium chromate (K 2CrO 4) and cadmium chloride (CdCl 2) was carried out in human blood lymphocytes in vitro as measured by the electron microscopy in situ end-labeling (EM-ISEL). EM-ISEL was used to assess DNA single-strand breaks (SSBs) expressed as number of immunogold particles per μm 2 of chromatin at both chromosomal and nuclear DNA levels. Human lymphocytes were cultured in supplemented RPMI medium for 72 h including treatment for 2 h with K 2CrO 4 (0–150 μM), CdCl 2 (0–150 μM) or methyl methanesulfonate (500 μM) as a positive control. Quantification of SSBs by EM-ISEL showed that both compounds are genotoxic agents at non-cytotoxic concentrations. This study brings new information on the utility of EM-ISEL for the evaluation of genotoxicity and confirms the genotoxic effects induced by chromium and cadmium.

Up-regulation and trafficking of δ opioid receptor in a model of chronic inflammation: implications for pain control

Pharmacological and physiological evidence supports a role for delta (δ) opioid receptors in the nociceptive mechanisms of inflammation. However, few data exist regarding δ opioid receptor expression and localization in such conditions. In this study, we have assessed the distribution and function of δ opioid receptors in the rat spinal cord following induction of chronic inflammation by intraplantar injection of complete Freund's adjuvant (CFA). Intrathecal administration of the selective δ opioid receptor agonist, d-[Ala 2, Glu 4] deltorphin, dose-dependently reversed thermal hyperalgesia induced by CFA. In situ hybridization and Western blotting experiments revealed an increase in δ opioid receptor mRNA and protein levels, respectively, in the dorsal lumbar spinal cord ipsilateral to the CFA injection site compared to the contralateral side and sham-injected controls. By electron microscopy, immunopositive δ opioid receptors were evident in neuronal perikarya, dendrites, unmyelinated axons and axon terminals. Quantification of immunopositive signal in dendrites revealed a twofold increase in the number of immunogold particles in the ipsilateral dorsal spinal cord of CFA-injected rats compared to the contralateral side and to sham-injected rats. Moreover, the relative frequency of immunogold particles associated with or in close proximity to the plasma membrane was increased in the ipsilateral dorsal spinal cord, indicating a more efficient targeting of δ opioid receptors to neuronal plasma membranes. These data demonstrate that CFA induces an up-regulation and increased membrane targeting of δ opioid receptors in the dorsal spinal cord which may account for the enhanced antinociceptive effects of δ opioid receptor agonists in chronic inflammatory pain models.

Altering expression of α3β1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin α3β1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of α3β1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin α3 subunit. The results showed that both the number of immunogold particles and the staining of integrin α3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of α3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the α3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin α3β1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.

Differential synaptic distribution of the AMPA-GluR2 subunit on GABAergic and non-GABAergic neurons in the basolateral amygdala

The cellular and ultrastructural distribution patterns of the AMPA glutamate receptor subunit, GluR2, were determined in the rat basolateral amygdala. GluR2 immunoreactivity was widely and uniformly distributed in the basolateral nucleus, with both pyramidal and non-pyramidal neurons labelled. In fact, double label immunohistochemical analyses demonstrated that over 90% of the GABAergic interneurons were labelled for GluR2. Electron microscopic analyses further confirmed the presence of GluR2 in the soma and dendrites of GABAergic interneurons as well as in the soma, spines and dendritic shafts of pyramidal cells. As in our parallel study in the rat hippocampus, immunogold analyses revealed that GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts, as well as the dendritic processes and soma of GABAergic interneurons. However, the number of immunogold particles per labelled synapse on GABAergic neurons was significantly lower than at similar labelled asymmetric synapses on spines of presumed pyramidal cells. Given that the presence of GluR2 within the AMPA receptor complex decreases calcium flux, these data indicate that GABAergic local circuit neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells, as has been suggested for hippocampus. Such cell class-specific differences in the subunit representation and resultant channel properties of AMPA receptors have implications for response properties as well as selective vulnerability of neurons within the basolateral nucleus of the amygdala.

Characterization of the 70-kDa Peroxisomal Membrane Protein, an ATP Binding Cassette Transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.

Immunocytochemical electron microscopic study and western blot analysis of myosin, paramyosin and miniparamyosin in the striated muscle of the fruit fly Drosophila melanogaster and in obliquely striated and smooth muscles of the earthworm Eisenia foetida.

Miniparamyosin is a paramyosin isoform (55-60 kDa) that has been isolated in insects (Drosophila) and immunolocalized in several species of arthropods, molluscs, annelids and nematodes. In this study, the presence and distribution of this protein, in comparison with that of paramyosin and myosin, has been examined in the striated muscle (tergal depressor of trochanter) of Drosophila melanogaster, and the obliquely striated muscle (body wall) and the smooth muscle (outer layer of the pseudoheart) of the earthworm Eisenia foetida by means of immunocytochemical electron microscopic study and Western blot analysis miniparamyosin paramyosin and myosin antibodies from Drosophila. In the striated muscle of D. melanogaster, the three proteins were immunolocalized along the length of the thick filaments (A-bands). The distribution of immunogold particles along these filaments was uniform. The relative proportions miniparamyosin/paramyosin/myosin (calculated by counting the number of immunogold particles) were: 1/10/68. In the obliquely striated muscle of E. foetida, immunoreactions to the three proteins were also found in the thick filaments, and the relative proportions miniparamyosin/paramyosin/myosin were 1/2.4/6.9. However, whereas the distribution of both myosin and miniparamyosin along the thick filament length was uniform, paramyosin immunolabelling was more abundant in the extremes of thick filaments (the outer zones of A-bands in the obliquely striated muscle), where the thick filaments become thinner than in the centre (the central zone of A-bands), where these filaments are thicker. The relative proportions of paramyosin in the outer and of paramyosin in the central zones of A-bands were 4/1. This irregular distribution of paramyosin along the thick filament length might be actual but it may also be explained by the fusiform shape of thick filaments in the earthworm: assuming that paramyosin is covered by myosin, paramyosin antigens would be more exposed in the tips than in the centre of thick filaments. If miniparamyosin is, in turn, covered by paramyosin, the exposure of miniparamyosin antigens would be low even in the tips of thick filaments, and this might explain the scanty immunoreaction observed for this protein and the absence of a higher number of immunogold particles in the extremes of thick filaments. The distribution of the three proteins in the earthworm smooth muscle was as in the obliquely striated muscle, although the proportions miniparamyosin/paramyosin/myosin were 1/1.5/5.2; this is, immunoreactions to paramyosin and miniparamyosin were lower than in the obliquely striated muscle.

Immunohistochemical study and western blotting analysis of titin-like proteins in the striated muscle of Drosophila melanogaster and in the striated and smooth muscle of the oligochaete Eisenia foetida.

The presence and distribution of titin-like proteins have been examined in transversely striated muscle of Drosophila melanogaster, in obliquely striated muscles (body wall and inner muscular layer of the pseudoheart) and smooth muscle (outer muscular layer of the pseudoheart) from the earthworm Eisenia foetida by means of Western blotting analysis, light microscopy immunohistochemistry, and electron microscopy immunogold labeling, using antibodies anti vertebrate (chicken) titin (3,000 kDa) and arthropod (D. melanogaster) mini-titin (twitchin or projectin) (700 kDa). To determine whether these antibodies immunoreact non-specifically against vertebrate titin, mouse skeletal muscle was also studied. As negative control, mouse smooth muscle was used. Immunoreaction to mini-titin was found in all the invertebrate muscles studied. For each of these muscles, Western blotting analysis of mini-titin showed a single band, at approximately 700 kDa. Electron microscopy immunolabeling to this protein was observed along the whole sarcomere length (A bands and I bands) in both transversely striated muscles of the insect and obliquely striated muscles of the earthworm, although the number of immunogold particles was more abundant in the insect muscles. Mini-titin immunolabeling was also observed in the smooth muscle cells that formed the outer layer of the earthworm pseudoheart although in lower amounts than in the obliquely striated muscle. The absence of true sarcomeres in the smooth muscle cells did not permit to determine the extension of mini-titin immunolabeling. No immunoreaction to this protein was found in the striated and smooth muscles of the mouse. Immunoreaction to titin was only observed in the mouse skeletal muscle, in which both A bands and I bands appeared immunolabeled. Present results show that mini-titin in the invertebrate muscles studied differs immunohistochemically from vertebrate titin and, in contrast with titin, mini-titin is also present in invertebrate smooth muscles.

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive subcanalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per micron of plasma membrane were 7.86 vs 2.46 (P less than 0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P less than 0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.